[Urinalysis: what a primary care physician needs to know]. / Analyse d'urines: l'ABC du praticien.

Dipstick urinalysis is a very useful diagnostic tool in primary care when used in a specific context (urinary complaints, follow-up of systemic diseases, or pregnancy), but not as a screening instrument. Urine collection in appropriate conditions, together with a correct interpretation of dipstick results, reduces the use of microscopic urinalysis and urine culture. Leucocyturia and positive nitrits indicate the presence of a urinary tract infection and do not generally require additional tests. Persistent haematuria or proteinuria need to be further explored (microscopic urinalysis and 24h urine collection). Presence of crystals in the microscopic urinalysis reflects the precipitation of the substance eliminated in the urinary tract, but does not systematically indicate a disease.

Detection of Pneumocystis jirovecii by two staining methods and two quantitative PCR assays.

BACKGROUND: Pneumocystis jirovecii is an opportunistic pathogen that causes pneumonia, particularly in immunodeficient hosts. MATERIALS AND METHODS: We retrospectively compared the results obtained by two staining methods (toluidine blue and calcofluor white) and two quantitative (q) real time PCR assays for the detection of P. jirovecii in bronchoalveolar lavage (BAL) specimens. For the qPCR assays, we used newly selected probes and primers targeting the Kex-1 gene, which codes for a serine endoprotease, and compared the results to those from the published assay targeting the beta-tubulin gene. RESULTS: A total of 1,843 BAL specimens were analyzed microscopically in parallel, and 74 (4.0%) were found to be positive with both stains, 23 (1.2%) were positive only with the toluidine blue stain, and six (0.3%) only with the calcofluor stain (p = 0.003). Of these, a selection of 186 consecutive BAL fluid samples were tested by qPCR using the respective different primer pairs. 21 of the 186 samples (11.3%) were microscopically positive with both stains as well as qPCR positive after 18-31 cycles (corresponding to 5.24 x 10(6) copies/ml to 640 copies/ml of native BAL) using the Kex-1 primer pair and between 21-33 cycles using the beta-tubulin assay. A good correlation between semi-quantitative microscopy and the number of PCR cycles needed for a positive signal was noted. Of the remaining 165 samples, 153 (82%) were both microscopically and PCR negative (PCR with the two sets of primers); the remaining 12 samples (7%) were Kex-1-based PCR positive (from cycles 33 to 41, corresponding to 160 copies/ml of BAL or less) but microscopically negative. Of these latter samples, ten (6%) were also positive (from cycles 34 to 38) with the primers targeting the beta-tubulin gene. Taking microscopy as a reference, the sensitivity of qPCR targeting the Kex-1 gene was 100%, and the specificity was 92.4%. CONCLUSION: The sensitive qPCR analysis proved to be a rapid and reliable method to detect P. jirovecii in BAL.

Efavirenz-induced urolithiasis.

We describe the first case of efavirenz-induced urolithiasis in a 47-year-old HIV-positive patient. Urinary obstruction led to pyelonephritis and septic shock, requiring emergency ureteral catheterisation. The subsequent clinical course was favourable, allowing the patient's discharge on day 5. A 7 mm, radio-translucent, non-crystalline, beige stone was extracted during catheterisation. Stone analysis by Fourier transform infrared spectrometry, liquid chromatography and mass spectrometry revealed a stone composed of efavirenz (EFV) metabolites M4, M5, M8 (as described by Mutlib et al. in 1999) and approximately 50% of unspecified proteins. EFV is a non-nucleoside reverse transcriptase inhibitor introduced to European markets in 1999. It is principally metabolised by cytochrome P450 3A4 and 2B6. Of the dose, 14-34% is excreted in the urine, 1% as unchanged drug. The patient had been taking 600 mg EFV per day for 3 years. As EFV-induced urolithiasis has not been reported so far, we would like to draw the attention of the medical community to this potentially severe complication.

Optimization of the kinetics of cooling of kidneys: a pig model.

We determined the kinetics of cooling in multiple organ procurement for the kidney in a pig model. A biometric analysis by regression enables us to define the factors which influence the rate of temperature decrease: weight of the donor, average rate of perfusion and difference of temperature between the rectal temperature and the temperature of the perfusion solution at initiation of cooling. The description of the temperature as a function of time follows an exponential model of the type T(t) = T0. e(dt) where d is the rate of decrease. The rate of decrease varies according to the above factors. The cellular viability ratio (CVR), was correlated to the rate of cooling. The mean CVR was 91% (SD 4.95) when the rate of cooling was more than 1 degrees C/min. This was compared to 75% (SD 11.17) when the rate was less than 1 degrees C/min (p = 0.023). Our experience leads us to believe that the average cooling rate is frequently too low (<1 degrees C/min). This model can be used to predict and control the kinetics of cooling and may help to define the best way of cooling for future xenotransplantation.

Characterization of two genes encoding the Mycobacterium tuberculosis ribonucleotide reductase small subunit.

Two nrdF genes, nrdF1 and nrdF2, encoding the small subunit (R2) of ribonucleotide reductase (RR) from Mycobacterium tuberculosis have 71% identity at the amino acid level and are both highly homologous with Salmonella typhimurium R2F. The calculated molecular masses of R2-1 and R2-2 are 36,588 (322 amino acids [aa]) and 36,957 (324 aa) Da, respectively. Western blot analysis of crude M. tuberculosis extracts indicates that both R2s are expressed in vivo. Recombinant R2-2 is enzymatically active when assayed with pure recombinant M. tuberculosis R1 subunit. Both ATP and dATP are activators for CDP reduction up to 2 and 1 mM, respectively. The gene encoding M. tuberculosis R2-1, nrdF1, is not linked to nrdF2, nor is either gene linked to the gene encoding the large subunit, M. tuberculosis nrdE. The gene encoding MTP64 was found downstream from nrdF1, and the gene encoding alcohol dehydrogenase was found downstream from nrdF2. A nrdA(Ts) strain of E. coli (E101) could be complemented by simultaneous transformation with M. tuberculosis nrdE and nrdF2. An M. tuberculosis nrdF2 variant in which the codon for the catalytically necessary tyrosine was replaced by the phenylalanine codon did not complement E101 when cotransformed with M. tuberculosis nrdE. Similarly, M. tuberculosis nrdF1 and nrdE did not complement E101. Activity of recombinant M. tuberculosis RR was inhibited by incubating the enzyme with a peptide corresponding to the 7 C-terminal amino acid residues of the R2-2 subunit. M. tuberculosis is a species in which a nrdEF system appears to encode the biologically active species of RR and also the only bacterial species identified so far in which class I RR subunits are not arranged on an operon.

Kinetics of cellular viability in warm versus cold ischemia conditions of kidney preservation. A biometric study.

We have determined the kinetics of the cellular viability ratio (CVR), defined as the number of living cells over the total cell count, in pig kidneys using propidium iodide and fluorescein diacetate staining, as a function of time and preservation conditions. The kidneys were preserved in warm or cold ischemia in order to mimic the conditions of transplantation from non-heart-beating donors or multiple removal with optimal preservation of the graft, respectively. To determine the CVR, the cells were obtained by a fine-needle aspiration biopsy, which minimizes the damage to the graft. A biometric analysis by regression enabled the determination of the time dependence for warm ischemia (CVR(t) = 80.0 x e(-0.733-t)(+2.7/-0.36)) and for cold ischemia (CVR(t) = 80.0 x e(-0.022-t)(+1.57/-0.64)) with a confidence interval of 95%. These master curves allow us to predict, under the described conditions, the CVR after a given ischemia time. The half-life of the cells can be deduced from the time-dependent CVR(t), and is 0.64 hr (38 min) for warm ischemia and 21.4 hr for cold ischemia. Further, the CVR for a given kidney can be used to assess its condition at removal: if the CVR is below 48% at 2 hr after removal, one can conclude that the organ has suffered a period of warm ischemia.

MRI findings in Caroli's disease and intrahepatic pigmented calculi.

This paper reports a case of Caroli's disease confined to the left lobe of the liver that mimicked left portal vein thrombosis on MRI studies because of the very high signal intensity on T1-weighted images of intrahepatic pigmented calculi. The preoperative diagnosis was a cholangiocarcinoma infiltrating the left hepatic bile duct and portal branch. The final macroscopic and histological diagnosis was Caroli's disease of the left liver lobe with wide enlarged left bile duct containing multiple pigmented calculi.

[What is the practical attitude toward isolated microscopic hematuria?]. / Quelle attitude pratique face à une hématurie microscopique isolée?

Practical attitude towards isolated microscopic hematuria. The finding of isolated asymptomatic microhematuria usually raises questions about the need to perform further, invasive investigations. Phase contrast microscopy of the urine sediment is a sensitive, noninvasive method that provides information on the glomerular or non glomerular origin of hematuria, as well as on its grade. This analysis, however, must be performed by an experienced technician under standard conditions. The presence of dysmorphic (i.e. glomerular) erythrocytes indicates a glomerular disease if the count is higher than 10 erythrocytes per microliter but is considered physiological if the count is below this number. In these two cases, no further investigation will be undertaken if all criteria for isolated microhematuria are verified. Conversely, isomorphic erythrocytes reveal a non glomerular origin of hematuria, which may indicate a serious urological disease. In this case, further investigations (e.g. ultrasound, urine cytology) are recommended, taking into account the age of the patient.

Developmental expression of the creatine kinase isozyme system of Xenopus: maternally derived CK-IV isoform persists far beyond the degradation of its maternal mRNA and into the zygotic expression period.

The differential expression of the multilocus CK isozyme system throughout development of the two Xenopus species X. laevis and X. borealis was investigated. A cDNA containing the nearly complete coding sequence of the CK-IV subunit of X. laevis was isolated and sequenced. Early development of X. laevis proceeds with a stock of maternally derived CK-IV/IV isozyme. While the mRNA declines rapidly after fertilization and disappears before neurulation, maternal CK-IV/IV isozyme is active far beyond the onset of zygotic expression and is still detectable when tadpoles start feeding. Zygotic expression of CK-IV begins after neurulation, at stage 22/24, and seems to start simultaneously with that of another gene, CK-III. Modulation in the expression of these two genes and the appearance of two other isoforms, the CK-I and CK-II/III isozymes, take place during development in a tissue-specific manner. During metamorphosis, the CK phenotypes of eyes and skeletal musculature undergo additional changes. The final adult pattern only appears several weeks after metamorphosis. The presumed orthologous CK isozymes of X. borealis show a developmental profile similar to that of X. laevis, except that CK-II/II is equally present in oocytes and during early development, in addition to CK-IV/IV isozyme. These results show that the expression of each of the four CK genes of Xenopus is under differential developmental control.

Genetic mapping in Xenopus laevis: eight linkage groups established.

Inheritance of alleles at 29 electrophoretically detected protein loci and one pigment locus (albinism) was analyzed in Xenopus laevis by backcrossing multiply heterozygous individuals generated by intersubspecies hybridization. Pairwise linkage tests revealed eight classical linkage groups. These groups have been provisionally numbered from 1 to 8 in an arbitrarily chosen order. Linkage group 1 includes ALB-2 (albumin), ADH-1 (alcohol dehydrogenase), NP (nucleoside phosphorylase), and ap (periodic albinism). Linkage group 2 contains ALB-1 and ADH-2, and probably is homeologous to group 1. Linkage group 3 comprises PEP-B (peptidase B), MPI-1 (mannosephosphate isomerase), SORD (sorbitol dehydrogenase), and mIDH-2 (mitochondrial isocitrate dehydrogenase). Linkage group 4 contains GPI-1 (glucosephosphate isomerase) and EST-4 (esterase 4). Linkage group 5 contains GPI-2 and PEP-D (peptidase D). Linkage group 6 comprises ACP-3 (acid phosphatase), sME (cytosolic malic enzyme), and GLO-2 (glyoxalase). Linkage group 7 consists of sSOD-1 (cytosolic superoxide dismutase), GPD-2 (glycerol-3-phosphate dehydrogenase), mME (mitochondrial malic enzyme), and the sex determining locus. Linkage group 8 includes FH (fumarate hydratase) and TRF (transferrin). Recombination frequencies between linked loci showed differences related to the genomic constitution (parental subspecies) and to the sex of the heterozygous parent. Independent assortment was observed between the duplicate ALB loci. This is true for the duplicate ADH, GLO, and MPI loci as well, supporting the view that these genes have been duplicated as part of a genome duplication that occurred in the evolutionary history of X. laevis. Comparative analysis of genetic maps reveals a possible conservation of several linkages from the Xenopus genome to the human genome.

Sex linkage of malic enzyme in Xenopus laevis.

Genetic analysis of mME variants (mitochondrial malic enzyme, E.C. 1.1.1.40) in Xenopus laevis revealed sex linkage of the mMe locus and indicated a WZ/ZZ type of sex determination. Codominant mMe alleles occur on both W and Z chromosomes, with a recombination frequency of 6.1% +/- 1.5% between mMe and the sex-determining locus (or region).

Albumin evolution in polyploid species of the genus Xenopus.

Electrophoresis of serum from 21 Xenopus species and subspecies reveals variable numbers of albumin bands. The diploid X. tropicalis has one albumin, while the tetraploid species (laevis, borealis, muelleri, clivii, fraseri, epitropicalis) have two. The octoploid species (amieti, boumbaensis, wittei, vestitus, andrei) have two to three bands, and the dodecaploid X. ruwenzoriensis has three. The molecular weight of the Xenopus albumins varies from 68 kd (in the tropicalis group) to 74 kd. The subspecies of X. laevis possess two albumins of different molecular weights (70 and 74 kd), whereas most species have only 70-kd albumins. Peptide maps have been obtained from albumin electromorphs by limited proteolysis in sodium dodecyl sulfate (SDS) gels, using S. aureus V8 protease. The peptide patterns produced by electromorphs from the same tetraploid Xenopus species generally differ from each other, suggesting that the two albumin genes contain a substantial amount of structural differences. In addition, the peptide maps are diagnostic for most tetraploid species and for some subspecies of X. laevis as well. Proteolysis of albumins from most octoploid and dodecaploid species results in patterns which are very similar to the ones produced by the electromorphs from X. fraseri. The albumins of X. vestitus differ from those of the other octoploid species. X. andrei possesses two fraseri-type and one vestitus-type albumin, which indicates that it probably originated by allopolyploidy.

Genetic variation for superoxide dismutase level in Drosophila melanogaster.

We have studied genetic variation for levels of activity of the enzyme superoxide dismutase (SOD) in Drosophila melanogaster. We have constructed 34 lines homozygous for a given second and a given third chromosome derived from eight original lines; all lines were homozygous for the "fast" (F) allele of Sod. The variation in the relative levels of SOD CRM ranges from 1 to 1.6. The second chromosomes modify the SOD level, even though the structural Sod locus is in the third chromosome, and the specific effect of a given second chromosome depends on the particular third chromosome with which it is combined. This indicates that the variation in SOD content is controlled by polygenic modifiers present in the second (and in the third) chromosome. In addition to these trans-acting modifiers, we have isolated a cis-acting element (SodCA1) that reduces SOD CRM levels to 3.5% of a typical F/F homozygote. SodCA1 is either a mutation in a regulatory site closely linked to the structural locus or a change in the coding sequence affecting the rate of degradation of the enzyme.

Experimental gynogenesis provides evidence of hybridogenetic reproduction in the Rana esculenta complex.

The gynogenetic offspring of the hybrid frog Rana esculenta (R. ridibunda X R. lessonae) are exclusively of the ridibunda type. This is due to the premeiotic exclusion of the lessonae genome from the hybrid's germ cells.

Biochemical variation in the Rana esculenta complex: a new hybrid form related to Rana perezi and Rana ridibunda.

Investigations of the green frogs from western Europe for electrophoretic variations at 4 enzyme loci demonstrated a new form which must be considered as a hybrid between Rana ridibunda and R. perezi. Biochemical evidence supports the hypothesis that its reproduction is hybridogenetic, as it is for R. esculenta.